The purpose and scope of this project is subdivided in four specific aims that are detailed below: Specific Aim 1: Characterize HIV-1 Membrane Receptor Disposition and Trafficking The plethora of new data on membrane proteins, lipids and glycoconjugates is changing our general view of membrane structure and function. Some of the emerging concepts are that membranes are patchy, with segregated regions of structure and function, that lipid regions vary in thickness and composition, and that crowding and ectodomains affect lateral mobility, and that the cytoskeleton is intimately involved in membrane dynamics. Since HIV fusion involves a complex cascade of interactions of the envelope glycoprotein with two receptors, membrane organization plays an important role and that interfering with this may modulate entry. We have changed organization by modifying membrane levels of cholesterol, ceramides, or glycosphingolipids. Most of these treatments led to fusion inhibition by restricting CD4 mobility. Specific Aim 2: Characterize Intermediates in Viral Fusion Host cell surface CD4 interactions with HIV gp120-gp41 elicit conformational changes in gp120, exposing co-receptor (CCR5 or CXCR4) binding sites, and in gp41, exposing the C-terminal heptad repeat region (C-HR) and the leucine/isoleucine zipper region (N-HR). Co-receptor engagement by gp120 triggers a battery of additional conformational changes in HIV gp41 eventually resulting in the formation of the thermo-stable 6-helix bundle (viral hairpin), which drives the membrane merger and eventual fusion. Synthetic peptides corresponding to N-HR and C-HR sequences potently inhibit membrane fusion by binding to the N-HR and C-HR domains on the viral envelope glycoprotein thereby interfering with the formation of the viral hairpin. The kinetics of interference with HIV fusion by a variety of inhibitors provides a way to study intermediates of the fusion process. The HIV pre-hairpin conformation can also be accessed by priming HIV Env-expressing cells with sCD4 and testing the effects of inhibitors. Specific Aim 3: Development of Site-Directed Photosensitized Labeling as a Tool to Study Membrane Protein Interactions We have developed a photosensitized labeling methodology that involves binding of photoactivatable probes to membrane proteins and lipids following activation of these probes in situ by energy transfer from a variety of donor chromophores. In the current studies we have used the membrane bilayer specific probe iodonaphthylazide (INA). We have used this method to establish which proteins of the viral envelope penetrate the target cell membrane in the course of infection and thus identify proteins and membrane compartments that participate in viral fusion. This research has lead to a novel method of inactivation of viruses in which INA (or other hydrophobic photoinduced alkylating probe) is inserted into the lipid bilayer of the viral envelope and subsequently irradiated with UV light. The probe selectively binds to protein domains in the lipid bilayer while leaving the ectodomains of these proteins unaffected. In this way we could obtain complete inactivation of viral infectivity with preservation of the integrity of its antigenic epitopes. Specific AIM 4: Elucidate Mechanisms of HIV-1 gp41 Induced Apoptosis HIV infections cause a progressive and irreversible depletion of CD4+ T cells leading to immunodeficiency. Apoptosis has been suggested to be a major mechanism of HIV mediated T cell loss. However, the mechanism via which HIV mediates apoptosis in either infected or uninfected cells is not clear. HIV-1 Env is expressed on the surface of infected cells and can interact with CD4 on uninfected cells. It has therefore been suggested that Env may be a likely candidate for induction of apoptosis in bystander cells We have explored this kiss of death mechanism in an in vitro co-culture system